The central hypothesis to be investigated in the proposed work is that cytochrome P-450 generated N-acetyl-p-quinoneimine is the agent responsible for acetaminophen hepatotoxicity. Attempts to confirm this hypothesis through direct study of the reactive species by conventional chemical methods have met with serious difficulties, and the quinoneimine has never been reported to have been isolated. A convenient electrochemical device has been developed which makes it possible to produce large quantities of the quinoneimine in solution. This makes it possible to thoroughly study the reactivity of the quinoneimine with various nucleophilic agents which result in the production of known metabolties. Furthermore, the liquid chromatography instrumentation and methods already developed in our laboratory have been shown to provide the method of choice for the rapid and sensitive assay of such synthetic reaction mixtures, as well as biological fluids, and tissue homogenates (esp. microsomal preparations). A careful comparison of the products obtained synthetically with those found in liver preparations and in vivo should provide powerful evidence as to the involvement of N-acetyl-p-quinoneimine in acetaminophen metabolism and toxicity. The proposed work will provide information of a fundamental nature regarding the metabolism of one of the most important over-the-counter drugs. Furthermore the effort will provide data of value in the design of antidotes for treatment of acetaminophen overdose cases. As an additional benefit, this investigation will serve as a model for study of other easily oxidized toxic amines and phenols such as benzidines, phenylenediamines, chloroanilines, chlorophenols, and diethylstilbestrol. The systematic combination of electrochemical, liquid chromatography, and stopped-flow spectroscopic studies affords a new approach to metabolism work which provides data often inaccessible by classical methodologies.